The polarity of epithelial cells is key to the function of adult epithelial organs such as kidney liver, intestine and exocrine glands. The hallmark of epithelial cell polarity is the asymmetric distribution of structural features and functional markers between the apical and basolateral domains of the plasma membrane. Our laboratory is interested in elucidating the molecular bases of this asymmetric distribution. Previous work from our group has demonstrated a key role for the trans Golgi network (TGN) in the sorting of these proteins into different vesicular pathways and identified cytoplasmic signals that mediate basolateral sorting of neural cell adhesion molecule (NCAM) and of a mutant form of nerve growth factor receptor (NGFR). A major goal of this proposal is to search for components of the protein machinery that bind to these signals and mediate incorporation of proteins into basolateral carrier vesicles. We will utilize a recently developed in vitro assay that measures the production of apical and basolateral carrier vesicles from the TON to characterize the role of ADP ribosylation factor (ARF) coatomer proteins (COPs), clathrin, adaptor proteins and G proteins in the production of apical and basolateral vesicles. The inhibitory role of basolateral signal peptides on basolateral vesicle release will be studied for different basolateral signals and basolateral proteins to characterize the pathways from the TON to the basolateral membrane. Replication defective adenovirus vectors will be used to introduce simultaneously apical and basolateral proteins in varying relative amounts to study the efficiency of sorting as a function of their expression levels. Components of the protein sorting machinery will be identified by their ability to bind cognate basolateral signal peptides as detected by blotting affinity chromatography and crosslinking with bifunctional reagents. The basolateral signal sequences of NCAM (39 amino acids) and a seven transmembrane segment receptor, the thyrotropin releasing hormone receptor (TRHR) will be characterized by site-directed mutagenesis and by bidimensional NMR (nuclear magnetic resonance). Subdomains of the extracellular portion of NCAM carrying apical sorting information will be identified by systematic subdomain deletion. Finally, we will search for proteins (v-SNARES, t-SNARES) that mediate specific docking of apical and basolateral vesicles to the respective cell surface. It is expected that these results will contribute important information to understand the processes of epithelial cell morphogenesis and differentiation.